The goal of these investigations is to understand the factors which control the folding of immunoglobulin subunits and their assembly into functional antibodies. Two human immunoglobulins are studied: IgG(Fro), an IgGl protein with k light chains and IgG(Gar), and IgG2 immunoglobulin with lambda light chains. The latter protein has a very high affinity for riboflavin and occurs in the serum with bound riboflavin. The protein offers an unusual opportunity to study assembly in the presence of a tightly bound hapten. Studies are projected also on two well-characterized mouse immunoglobulins, MOPC-31c and MPC-11, each with distinctive in vivo assembly patterns and kinetics. In addition, assembly variants of MPC-11 arising in mutagenized clones will be examined for in vitro assembly characteristics. Attempts will also be made to investigate the non-covalent and covalent assembly of hybrid mouse immunoglobulins prepared by oxidation of subunits from different proteins and in different subclasses. In all of these studies, the in vitro pathways and kinetics of non-convalent and covalent assembly will be examined, as will the identification of intermediates and the kinetics of conversion of each intermediate on the pathway. The influence of L chains on rates of disulfide formation between H chains, and of H chains on rates of formation of the H-L half molecule will be investigated by oxidations of separated H chains in the presence and absence of L chains, and by oxidation of HL half molecules, in the absence of free H or L chains, respectively. The influence of domain interactions on folding and assembly will be studied by examining rates and pathways of non-covalent and covalent assembly using separated C and V region peptides, mixtures of the peptides, and modified light chains. Various spectral methods will be employed as probes of conformation of the separated and recombined fragments, and rapid mixing techniques, including newly devised for UV stopped-flow CD measurements, will be employed to investigate the kinetics of chain folding and association.